Our objective is to identify and characterize all of the molecular components necessary for in vitro DNA replication. Our own work suggests that this approach might be feasible for eukaryotic DNA replication. The immediate objective of this research proposal is to establish a multi-enzyme system for DNA replication using Xenopus laevis proteins in vitro, and to implicate these proteins in embryonic DNA replication in vivo. The mid-range objective of this research is to pinpoint the molecules controlling nuclear DNA replication in various regions of the embryo during embryogenesis. The long-range objective of this research is to formulate general principles governing morphogenesis in molecular terms. At present, we are the only laboratory to have purified the eight or so proteins likely to be involved in DNA replication from a single eukaryotic organis, and we are fortunate to have a series of well-defined DNA molecules (i.e. containing "natural" origins of DNA replication) from the same organism. The methods involved conventional procedures of enzyme purification to obtain electrophoretically homogeneous preparations of molecules involved in DNA replication for the study of in vitro DNA synthesis in reconstituted systems. In vivo DNA replication will be studied by microinjection of labeled precursors. Detection and characterization of replicating DNA molecules will be carried out by electron microscopy and electron microscope, autoradiography as well as conventional label incorporation and hydrodynamic methods.